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Abstract Shiga toxin-producing Escherichia coli (STEC) strains have emerged as important foodborne pathogens of global public health concern, causing life-threatening diseases. Sheep and their products have been documented as important reservoirs for STECs, especially E. coli O157. The aim of this study was to investigate STECs from diarrheal human and sheep in Al-Madinah Al-Munawarah, Saudi Arabia. Fecal samples were collected between June and August, 2015 from diarrheal humans (n = 134) and sheep (n = 87). Presumptive E. coli human-and sheep-isolated strains were identified for their serotypes, the associated virulence genes (Shiga toxin [stx1 , stx2 ], haemolysin [ehxA] and intimin [eae]) by polymerase chain reaction and their susceptibility to antibiotics. Pulsed-field gel electrophoresis (PFGE) was used to demonstrate the genetic relatedness between Serotype O157:H7 human- and sheep-isolated strains. Forty eight (48/221; 21.7%) STECs were recovered from both human and sheep, their serotypes were as follows: O157:H7, O26:H11, O157:HNM, O26:HNM, O128:H2, O48:HNM, O111:HNM and OUT:HUT. Various virulence profiles and multiple antibiotic resistance were observed among the isolates. Twenty eight O157:H7 serotypes (17 human isolates and 11 sheep isolates) were identified in 13 PFGE pulsotypes, where human and sheep isolates were highly related. PFGE banding profiles together with serotypes and genotypes afford proof that human and sheep can be colonized and infected with similar E. coli O157:H7 strains. Our findings highlight the importance of epidemiological and microbiological surveillance of STECs; as well as the development of control measures to decrease risks associated with zoonotic O157:H7.